WO2010011175A1 - Method of high frequency regeneration of sorghum - Google Patents
Method of high frequency regeneration of sorghum Download PDFInfo
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- WO2010011175A1 WO2010011175A1 PCT/SG2008/000266 SG2008000266W WO2010011175A1 WO 2010011175 A1 WO2010011175 A1 WO 2010011175A1 SG 2008000266 W SG2008000266 W SG 2008000266W WO 2010011175 A1 WO2010011175 A1 WO 2010011175A1
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G7/00—Botany in general
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G22/00—Cultivation of specific crops or plants not otherwise provided for
- A01G22/20—Cereals
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
Definitions
- the present invention relates generally to the regeneration of sorghum involving organogenesis. More specifically, the present invention relates to a method of regenerating sorghum, particularly Sorghum bicolor (L.) Moench, via organogenesis that yields a high frequency of regenerants.
- sorghum particularly Sorghum bicolor (L.) Moench
- Sorghum ⁇ Sorghum bicolor (L.) Moench) is a widely grown grain and forage crop, and more closely related to the major crops of tropical origin such as rice, maize, sugarcane, and pearl millet. Sorghum ranks fifth in production among cereal crops, and is an important model for tropical grasses. It is unique among major cereals because it adapts well to environmental extremes, notably drought and heat.
- sorghum the logical grain to support human and animal populations in areas with extreme heat and minimal precipitation (Howe et al. 2006).
- biofuel alternatives such as bioethanol and biodiesel.
- biofuels can be produced from a diverse set of crops, each country is adopting a strategy that exploits the comparative advantages it holds in certain crops.
- biotechnological techniques such as tissue culture and transformation can be utilized.
- the present invention relates generally to the regeneration of sorghum involving organogenesis. More specifically, the present invention relates to a method of regenerating sorghum, particularly Sorghum bicolor (L.) Moench, via organogenesis that yields a high frequency of regenerants.
- sorghum particularly Sorghum bicolor (L.) Moench
- the present invention provides for the first time a method for the high frequency regeneration of sweet sorghum ⁇ Sorghum bicolor (L.) Moench).
- sorghum is regenerated via organogenesis using dissected embryonated cotyledon (DEC) explants.
- DEC dissected embryonated cotyledon
- the present invention is widely applicable to sweet sorghum varieties and hybrids, and is specifically illustrated herein with respect to sweet sorghum ⁇ Sorghum bicolor (L.) Moench) hybrid varieties Liotian 1, Lioza 7050A and Liao Ji Za 1.
- the present invention can be applied directly to the production of sorghum variants through somoclonal variation and to the genetic transformation of sorghum.
- organogenic callus is induced from dissected embryonated cotyledon (DEC) explants of Chinese sweet sorghum ⁇ Sorghum bicolor (L.) Moench) and plants are regenerated from the organogenic callus.
- the high frequency regeneration system of the present invention consists of six-stages: stage 1 (callus induction), stage 2 and 3 (shoot bud induction and reduction of phenolic exudates), stage 4 (shoot bud induction and elongation), stage 5 (shoot elongation and reduction of phenolic exudates) and stage 6 (rooting).
- MS mineral salts supplemented with N6 vitamins in stage 1 MS mineral salts supplemented with B5 vitamins in the subsequent stages and the alteration of antioxidants (e.g., polyvinyl pyrrolidone (PVP) and polyvinyl polypyrrolidone (PVPP)), organic additives (e.g., adenine sulfate (AdSO 4 )), inorganic additives (e.g., AgNO 3 ), amino acids (e.g., L-proline, L- glutamine, L-glycine) and PGRs (plant growth regulators) at specific stages enhances the efficiency of regeneration and reduces the exudation of high phenolic compounds inherent in sorghum tissue culture. Approximately 250-300 healthy plantlets devoid of any significant variation in genotypes are produced from a single DEC explant within a short span of three months time with 100% survivability.
- PVP polyvinyl pyrrolidone
- PVPP polyvinyl polypyrrolidone
- Figures IA- II illustrate the regeneration of sorghum via organogenesis from dissected embryonated cotyledons (DEC) in accordance with the present invention.
- Figures IA and IB show dissected embryonated cotyledon explants without radicals and callus induction in and around the cotyledonary node.
- Figure 1C shows shoot bud induction from the DEC callus.
- Figures ID and IE show multiple shoot regeneration from shoot buds.
- Figure IF shows elongation of the regenerated shoots.
- Figures G and H show rooting of the elongated shoots.
- Figure II shows hardened plantlets.
- the present invention relates generally to the regeneration of sorghum involving organogenesis. More specifically, the present invention relates to a method of regenerating sorghum, particularly Sorghum bicolor (L.) Moench, via organogenesis that yields a high frequency of regenerants. In addition to providing high frequency regeneration, the present invention can be applied directly to the production of sorghum variants through somoclonal variation and to the genetic transformation of sorghum.
- organogenesis refers to the regeneration of shoots either via callus or direct meristematic tissues of any part of plants in a specific medium under controled environmental conditions.
- callus refers to an unorganized or undifferentiated mass of proliferative cells produced from any explant tissue.
- a method for regenerating sorghum through organogenesis that efficiently yields a high frequency of regenerated plants.
- plants are regenerated from explants obtained from embryonated cotyledon explants of sorghum, particularly Sorghum bicolor (L.) Moench.
- the embryonated cotyledon explants are dissected and placed on a solid medium, referred to as stage 1 medium herein.
- the stage 1 medium comprises MS mineral salts and N6 vitamins and is supplemented with L-proline and L-glutamine.
- the stage 1 medium is supplemented with about 25 mg to about 200 mg, preferably about 50 mg to about 100 mg, more preferably about 100 mg L-proline. In another embodiment, the stage 1 medium is supplemented with about 50 mg to about 250 mg, preferably about 50 mg to about 100 mg, more preferably about 100 mg L-glutamine.
- the stage 1 medium also contains plant growth regulators.
- the plant growth regulators are an auxin and abscisic acid (ABA).
- the auxin is 2,4-dichlorophenoxy acetic acid (2,4-D).
- the concentration of ABA in the stage 1 medium is about 0.94 ⁇ M to about 18.9 ⁇ M, preferably about 0.94 ⁇ M to about 7.56 ⁇ M, more preferably about 3.78 ⁇ M.
- the concentration of 2,4-D in the stage 1 medium is about 2.26 ⁇ M to about 9.04 ⁇ M, preferably about 4.52 ⁇ M to about 6.78 ⁇ M, more preferably about 6.78 ⁇ M.
- the stage 1 medium also contains a source of carbon, hi one embodiment, the source of carbon is sucrose at about 2% to about 5%, preferably about 2% to about 3%, more preferably, about 3%. In another embodiment, the source of carbon is glucose at about 2% to about 5%, preferably about 3% to about 5%.
- the source of carbon is fructose at about 2% to about 5%, preferably about 2% to about 3%. In a further embodiment, the source of carbon is maltose at about 2% to about 5%, preferably about 2% to about 3%. In another embodiment, the source of carbon is a mixture of sucrose and glucose at about 2% to about 3%, preferably about 2% sucrose and about 2% to about 3%, preferably about 2% glucose. In an additional embodiment, the source of carbon is a mixture of fructose and glucose at about 1% to about 2%, preferably about 1% fructose and about 2% to about 3%, preferably about 2% glucose. In a further embodiment, the source of carbon is a mixture of maltose and glucose at about 1% to about 2%, preferably about 1% maltose and about 2% to about 3%, preferably about 2% glucose.
- the DEC segments are cultured on the stage 1 medium for about four weeks to about eight weeks, preferably about six weeks.
- the stage 1 cultures are subcultured at about two week intervals.
- the stage 1 culture is maintained at about 25° C ⁇ 2° C with a 16h/8h (light/dark) photoperiod at 55% to 60% relative humidity.
- the light photoperiod is under white fluorescent lights with a light intensity of about 25 ⁇ E m " s " .
- the DEC segments free of radicals cultured on stage 1 medium show suppressed growth, but at the same time the explants became swollen, stumped and yellowish green.
- SCPC cotyledonary node
- the radical tissues also started to bulge within two weeks. These radical tissues were completely removed from the explants.
- stage 2 medium comprises MS mineral salts and B5 vitamins and is supplemented with L-proline, L-glutamine, L-glycine, ascorbic acid (Asc), AgNO 3 , PVP and PVPP.
- the stage 2 medium is supplemented with about 100 mg to about 1000 mg, preferably about 300 mg to about 600 mg, more preferably about 500 mg L-proline.
- the stage 2 medium is supplemented with about 100 mg to about 1000 mg, preferably about 300 mg to about 600 mg, more preferably about 500 mg L-glutamine.
- the stage 2 medium is supplemented with Asc at a concentration of about 1.42 ⁇ M to about 11.36 ⁇ M, preferably about 2.84 ⁇ M to about 8.52 ⁇ M, more preferably about 5.67 ⁇ M.
- the stage 2 medium is supplemented with about 0.25 g to about 1.5 g, preferably about 0.5 g to about 1.5 g, more preferably about 1.0 g PVP.
- the stage 2 medium is supplemented with about 0.25 g to about 1.5 g, preferably about 0.5 g to about 1.0 g, more preferably about 1.0 g PVPP.
- the stage 2 medium is supplemented with about 2 mg to about 20 mg, preferably about 5 mg to about 15 mg, more preferably about 10 mg L-glycine. In another embodiment, the stage 2 medium is supplemented with about 0.2 mg to about 1.5 mg, preferably about 0.5 mg to about 1.0 mg, more preferably about 1.0 mg AgNO 3 .
- the stage 2 medium also contains a plant growth regulator.
- the plant growth regulator is an auxin.
- the auxin is (2,4-D.
- the concentration of 2,4-D in the stage 2 medium is about 2.26 ⁇ M to about 9.04 ⁇ M, preferably about 4.52 ⁇ M to about 6.78 ⁇ M, more preferably about 6.78 ⁇ M.
- the stage 2 medium also contains a source of carbon.
- the source of carbon in the stage 2 medium may be the same as in the stage 1 medium.
- the source of carbon is sucrose at about 2% to about 5%, preferably about 2% to about 3%, more preferably, about 3%.
- the injured SCPC is cultured on the stage 2 medium for about three days to about seven days, preferably about five days.
- the stage 2 culture is maintained at about 25° C ⁇ 2° C with a 16h/8h (light/dark) photoperiod at 55% to 60% relative humidity as described above.
- shoot buds started to induce from the plumular callus as well as in and around the cotyledonary node.
- enormous phenolic exudates started oozing out from the base of the shoot buds.
- Asc, PVP, PVPP, L-glycine and AgNO 3 to the stage 2 medium prevented these phenolic exudates.
- Long time culture of SCPC more than five days in stage 2 medium inhibited the developing shoot buds which simultaneously, became brown and ultimately senesced.
- stage 3 medium comprises MS mineral salts and B5 vitamins and is supplemented with L-glutamine, L-glycine, casein hydrolysate (CH), adenine sulfate (AdSO 4 ) and PVP.
- the stage 3 medium is supplemented with about 100 mg to about 1000 mg, preferably about 300 mg to about 600 mg, more preferably about 500 mg L-glutamine.
- the stage 3 medium is supplemented with about 5 mg to about 25 mg, preferably about 10 mg to about 20 mg, more preferably about 20 mg L-glycine.
- the stage 3 medium is supplemented with about 0.25 g to about 1.5 g, preferably about 0.5 g to about 1.5 g, more preferably about 1.0 g PVP. In one embodiment, the stage 3 medium is supplemented with about 0.5 g to about 1.5 g, preferably about 0.5 g to about 1.0 g, more preferably about 1.0 g CH. In another embodiment, the stage 3 medium is supplemented with about 25 mg to about 200 mg, preferably about 50 mg to about 100 mg, more preferably about 100 mg AdSO 4 .
- the stage 3 medium also contains plant growth regulators. In one embodiment, the plant growth regulators are auxins and cytokinins. In a preferred embodiment, the auxin is 2,4-D.
- the concentration of 2,4-D in the stage 3 medium is about 0.56 ⁇ M to about 2.24 ⁇ M, preferably about 0.56 ⁇ M to about 1.12 ⁇ M, more preferably about 1.12 ⁇ M.
- the cytokinins are a mixture of 6-benzylaminopurine (BA) and kinetin (KN).
- the concentration of BA in the stage 3 medium is about 4.43 ⁇ M to about 13.29 ⁇ M, preferably about 4.43 ⁇ M to about 11.07 ⁇ M, more preferably about 8.86 ⁇ M.
- the concentration of KN in the stage 3 medium is about 2.32 ⁇ M to about 18.56 ⁇ M, preferably about 4.64 ⁇ M to about 13.92 ⁇ M, more preferably about 9.28 ⁇ M.
- the stage 3 medium also contains a source of carbon.
- the source of carbon in the stage 3 medium may be the same as in the stage 1 medium.
- the source of carbon is sucrose at about 2% to about 5%, preferably about 2% to about 3%, more preferably, about 3%.
- the tissue is cultured on the stage 3 medium for about seven days to about 15 days, preferably about ten days.
- the stage 3 cultures are subcultured at about five day intervals.
- the stage 3 culture is maintained at about 25° C ⁇ 2° C with a 16h/8h (light/dark) photoperiod at 55% to 60% relative humidity as described above.
- auxin (2,4-D) concentration was reduced, cytokinins, (BA, KN) and organic additives (such as CH, AdSO 4 , PVP) were added to enhance shoot bud proliferation and growth.
- stage 4 medium After culturing on the stage 3 medium, the proliferating shoot buds from a single clump are split into 8-10 clumps with each clump containing three to five shoot buds and cultured on a solid medium referred to as stage 4 medium herein.
- the stage 4 medium comprises MS mineral salts and B5 vitamins and is supplemented with L-glycine and adenine sulfate (AdSO 4 ).
- the stage 4 medium is supplemented with about 5 mg to about 25 mg, preferably about 10 mg to about 20 mg, more preferably about 20 mg L- glycine.
- the stage 4 medium is supplemented with about 25 mg to about 200 mg, preferably about 50 mg to about 100 mg, more preferably about 100 mg AdSO 4 .
- the stage 4 medium also contains plant growth regulators.
- the plant growth regulators are a cytokinin and gibberellic acid (GA 3 ).
- the cytokinin is BA.
- the concentration of BA in the stage 4 medium is about 4.43 ⁇ M to about 13.29 ⁇ M, preferably about 4.43 ⁇ M to about 11.07 ⁇ M, more preferably about 8.86 ⁇ M.
- the concentration of GA 3 in the stage 4 medium is about 0.72 ⁇ M to about 2.88 ⁇ M, preferably about 0.72 ⁇ M to about 2.16 ⁇ M, more preferably about 1.44 ⁇ M.
- the stage 4 medium also contains a source of carbon.
- the source of carbon in the stage 4 medium may be the same as in the stage 1 medium.
- the source of carbon is sucrose at about 2% to about 5%, preferably about 2% to about 3%, more preferably, about 3%.
- the proliferating shoot buds are cultured on the stage 4 medium for about one week to about three weeks, preferably about two weeks.
- the stage 4 cultures are subcultured at about one week intervals.
- the stage 4 culture is maintained at about 25° C ⁇ 2° C with a 16h/8h (light/dark) photoperiod at 55% to 60% relative humidity as described above. After two weeks about 25-30 shoot buds with shoot regeneration occurred from each single clump.
- Plant growth regulators (PRGs) (2,4-D and KN) and organic additives (PVP 5 CH, L-glutamine) were removed and GA 3 were supplemented for shoot elongation in stage 4.
- stage 5 medium comprises MS mineral salts and B5 vitamins and is supplemented with L-glycine, PVP and adenine sulfate (AdSO 4 ).
- the stage 5 medium is supplemented with about 5 mg to about 25 mg, preferably about 10 mg to about 20 mg, more preferably about 20 mg L- glycine.
- the stage 5 medium is supplemented with about 25 mg to about 200 mg, preferably about 50 mg to about 100 mg, more preferably about 100 mg AdSO 4 .
- the stage 5 medium is supplemented with about 0.5 g to about 1.5 g, preferably about 0.5 g to about 1.0 g, more preferably 1.0 g PVP.
- the stage 5 medium also contains plant growth regulators.
- the plant growth regulators are a cytokinin and gibberellic acid (GA 3 ).
- the cytokinin is BA.
- the concentration of BA in the stage 5 medium is about 4.43 ⁇ M to about 13.29 ⁇ M, preferably about 4.43 ⁇ M to about 11.07 ⁇ M, more preferably about 8.86 ⁇ M.
- the concentration of GA 3 in the stage 5 medium is about 0.72 ⁇ M to about 2.88 ⁇ M, preferably about 0.72 ⁇ M to about 2.16 ⁇ M, more preferably about 1.44 ⁇ M.
- the stage 5 medium also contains a source of carbon.
- the source of carbon in the stage 5 medium may be the same as in the stage 1 medium.
- the source of carbon is sucrose at about 2% to about 5%, preferably about 2% to about 3%, more preferably, about 3%.
- the proliferating shoot buds are cultured on the stage 5 medium for about one week to about three weeks, preferably about two weeks.
- the stage 5 cultures are subcultured at about one week intervals.
- the stage 5 culture is maintained at about 25° C ⁇ 2° C with a 16h/8h (light/dark) photoperiod at 55% to 60% relative humidity as described above.
- PVP is added to the stage 5 medium as an anti-oxidizing agent. The PVP does not inhibit the shoot growth but enhances shoot proliferation. The shoot proliferation and growth was maximum (4 cm) in stage 5 medium within two weeks.
- stage 6 medium After culturing on the stage 5 medium, the shoot clumps with 2-3 shoots of approximately 4 cm in height are transferred to a solid medium referred to as stage 6 medium herein for rooting.
- the stage 6 medium comprises MS mineral salts and B 5 vitamins and is supplemented with L-glycine, PVP and adenine sulfate (AdSO 4 ).
- the stage 6 medium is supplemented with about 5 mg to about 25 mg, preferably about 10 mg to about 20 mg, more preferably about 20 mg L-glycine.
- the stage 6 medium is supplemented with about 25 mg to about 200 mg, preferably about 50 mg to about 100 mg, more preferably about 100 mg AdSO 4 .
- the stage 6 medium is supplemented with about 0.5 g to about 1.5 g, preferably about 0.5 g to about 1.0 g, more preferably 1.0 g PVP.
- the stage 6 medium also contains a plant growth regulator.
- the plant growth regulator is an auxin.
- the auxin is indole 3-acetic acid (IAA).
- the concentration of IAA in the stage 6 medium is about 2.85 ⁇ M to about 11.4 ⁇ M, preferably about 2.85 ⁇ M to about 8.55 ⁇ M, more preferably about 5.7 ⁇ M.
- the source of carbon in the stage 6 medium may be the same as in the stage 1 medium. In a preferred embodiment, the source of carbon is sucrose at about 2% to about 5%, preferably about 2% to about 3%, more preferably, about 3%.
- the proliferating shoot buds are cultured on the stage 6 medium for about one week to about three weeks, preferably about two weeks.
- the stage 6 culture is maintained at about 25° C ⁇ 2° C with a 16h/8h (light/dark) photoperiod at 55% to 60% relative humidity as described above.
- the auxin, IAA in stage 6 medium induced roots within one week. There was no root formation on plant growth regulator free medium or medium containing only cytokinins. Auxin was essential for root induction and without auxin, there was no root formation. An average of 10-15 roots of 3 cm long are produced per shoot when cultured in stage 6 medium.
- the rooted plantlets are successfully hardened with about 100 % survivability and transferred to the green house for further growth.
- the present invention provides systems which can be used for the in transformation of sorghum plants so that high frequency of transgenic plants can be obtained.
- the method of transformation/transfection is not critical to the transformation of sorghum plants; various methods of transformation or transfection are currently available. As newer methods are available to transform crops or other host cells they may be directly applied. Accordingly, a wide variety of methods have been developed to insert a DNA sequence into the genome of a host cell to obtain the transcription and/or translation of the sequence to effect phenotypic changes in the organism. Thus, any method, which provides for effective transformation/transfection may be employed. See, for example, Mathews et al. ( 1992), Neuhaus et al. (1987), Wilde et al.
- the explant tissue can be co-cultured with an Agrobacterium strain harboring one or more DNA constructs containing one or more genes or nucleic acids of interest using techniques well known in the art.
- Transformed tissue can be selected using conventional techniques well known in the art.
- the DNA can be introduced into the explant tissue using conventional techniques, such as particle bombardment.
- Transformed tissue can be selected using conventional techniques well known in the art.
- Transformed or transgenic plants can be regenerated using the methods described herein.
- the DNA that is inserted (the DNA of interest) into sorghum plants is not critical to the transformation process. Generally the DNA that is introduced into a plant is part of a construct.
- the DNA may be a gene of interest, e.g., a coding sequence for a protein, or it may be a sequence that is capable of regulating expression of a gene, such as an antisense sequence, a sense suppression sequence or a miRNA sequence.
- the construct typically includes regulatory regions operatively linked to the 5' side of the DNA of interest and/or to the 3' side of the DNA of interest. A cassette containing all of these elements is also referred to herein as an expression cassette.
- the expression cassettes may additionally contain 5' leader sequences in the expression cassette construct.
- the regulatory regions (i.e., promoters, transcriptional regulatory regions, and translational termination regions) and/or the polynucleotide encoding a signal anchor may be native/analogous to the host cell or to each other.
- the regulatory regions and/or the polynucleotide encoding a signal anchor may be heterologous to the host cell or to each other. See, U.S. Patent No. 7,205,453 and U.S. Patent Application Publication Nos. 2006/0218670 and 2006/0248616.
- the expression cassette may additionally contain selectable marker genes. See, U.S. Patent No. 7,205,453 and U.S. Patent Application Publication Nos. 2006/0218670 and 2006/0248616.
- the expression cassette will comprise a selectable marker gene for the selection of transformed cells.
- Selectable marker genes are utilized for the selection of transformed cells or tissues.
- the plant selectable marker gene will encode antibiotic resistance, with suitable genes including at least one set of genes coding for resistance to the antibiotic spectinomycin, the streptomycin phosphotransferase (spt) gene coding for streptomycin resistance, the neomycin phosphotransferase (nptll) gene encoding kanamycin or geneticin resistance, the hygromycin phosphotransferase (hpt or aphiv) gene encoding resistance to hygromycin, acetolactate synthase (als) genes.
- the plant selectable marker gene will encode herbicide resistance such as resistance to the sulfonylurea-type herbicides, glufosinate, glyphosate, ammonium, bromoxynil, imidazolinones, and 2,4- dichlorophenoxyacetate (2,4-D), including genes coding for resistance to herbicides which act to inhibit the action of glutamine synthase such as phosphinothricin or basta (e.g., the bar gene). See generally, WO 02/36782, U.S. Patent No. 7,205,453 and U.S. Patent Application Publication Nos. 2006/0248616 and 2007/0143880, and those references cited therein.
- herbicide resistance such as resistance to the sulfonylurea-type herbicides, glufosinate, glyphosate, ammonium, bromoxynil, imidazolinones, and 2,4- dichlorophenoxyacetate (2,4-D
- selectable marker genes is not meant to be limiting. Any selectable marker gene can be used.
- a number of promoters can be used in the practice of the invention. The promoters can be selected based on the desired outcome. That is, the nucleic acids can be combined with constitutive, tissue-preferred, or other promoters for expression in the host cell of interest. Such constitutive promoters include, for example, the core promoter of the Rsyn7 (WO 99/48338 and U.S. Patent No.
- promoters include inducible promoters, particularly from a pathogen-inducible promoter.
- Such promoters include those from pathogenesis-related proteins (PR proteins), which are induced following infection by a pathogen; e.g., PR proteins, SAR proteins, beta-1,3- glucanase, chitinase, etc.
- PR proteins pathogenesis-related proteins
- Other promoters include those that are expressed locally at or near the site of pathogen infection.
- the promoter may be a wound-inducible promoter, hi other embodiments, chemical-regulated promoters can be used to modulate the expression of a gene in a plant through the application of an exogenous chemical regulator.
- the promoter may be a chemical-inducible promoter, where application of the chemical induces gene expression, or a chemical-repressible promoter, where application of the chemical represses gene expression.
- tissue-preferred promoters can be utilized to target enhanced expression of a polynucleotide of interest within a particular plant tissue. Each of these promoters is described in U.S. Patent Nos. 6,506,962, 6,575,814, 6,972,349 and 7,301,069 and in U.S. Patent Application Publication Nos. 2007/0061917 and 2007/0143880. [0032] Where appropriate, the DNA of interest may be optimized for increased expression in the transformed plant.
- the coding sequences can be synthesized using plant-preferred codons for improved expression.
- Methods are available in the art for synthesizing plant- preferred genes. See, for example, U.S. Patent Nos. 5,380,831, 5,436,391, and 7,205,453 and U.S. Patent Application Publication Nos. 2006/0218670 and 2006/0248616.
- RNA Interference Technology The Nuts & Bolts ofsiRNA Technology, DNA Press, 2003; Gott, RNA Interference, Editing, and Modification: Methods and Protocols (Methods in Molecular Biology), Human Press, Totowa, NJ, 2004; Sohail, Gene Silencing by RNA Interference: Technology and Application, CRC, 2004..
- Seeds of sweet sorghum (Sorghum bicolor (L.) Moench) hybrid varieties Liotian 1 , Lioza 7050A and Liao Ji Za 1 were obtained from Chinese National Center for Sorghum Improvement, Shenyang City, 110161, Liaoning City, P.R. China.
- the viable seeds were washed in chlorohexidine surgical wash (two drops in 100 ml of sterile water) for five minutes and soaked in sterile water for two days at 4° C in refrigerator. Later these imbibed seeds were further surface sterilized in 10% clorox solution (commercial bleach) for 10 minutes followed by washes with sterile water for five times. To ensure complete sterility, these seeds were rinsed again with 70% alcohol for 90 seconds and subsequently washed with sterile water three times before used as explants.
- 10% clorox solution commercial bleach
- the seed coats were removed from the sterilized seeds of all the three hybrid varieties (Liotian 1, Lioza 7050A and Liao Ji Za 1) and the embryonated cotyledon explants were dissected and inoculated aseptically on the stage 1 medium (Table 1) for swelling, callusing of plumules in and around the cotyledonary nodal regions (SCPC).
- the radical tissues were completely removed from the dissected embryonated cotyledon (DEC) explants. After six weeks of culture, the SCPC from stage 1 medium were further injured and transferred to stage 2 medium (Table 1) and stage 3 medium (Table 1) at five and ten-day interval for shoot bud induction.
- stage 3 medium the shoot buds developed on stage 3 medium were split into 8-10 clumps with each clumps containing three to five shoots and transferred to stage 4 medium (Table 1) for further shoot induction followed by stage 5 medium (Table 1) for shoot elongation at two-week intervals respectively.
- stage 4 medium stage 4 medium
- stage 5 medium stage 5 medium
- the elongated shoot clumps with 2-3 shoots (4cm height) from stage 5 medium were transferred to stage 6 medium (Table 1) for root induction.
- stage 6 medium stage 1 for root induction.
- the well developed and rooted plantlets were transferred for hardening after two weeks of culture.
- N6 vitamin Nitch and Nitch vitamins.
- 2,4-D 2,4-dichlorophenoxy acetic acid.
- B5 vitamin Gamborg vitamins.
- PVP polyvinylpyrrolidone
- PVPP polyvinylpolypyrrolidone
- BA or BAP 6-benzylamniopurine or benzyl adenine.
- KN kinetin.
- CH casein hydrolysate.
- AdSO 4 adenine sulfate.
- GA 3 g ⁇ bberellic acid.
- IAA indole 3-acetic acid.
- the pH of the medium was adjusted to 5.8 with IN NaOH or IN HCl prior to the addition of 0.8% phytoagar (stages 1-5 media) or 0.25% phytogel (stage 6 medium) and sterilized by autoclaving at 121 0 C for 20 minutes. Approximately 20 ml of sterilized medium was dispensed into sterilized Petri dish (90 X 15 mm size, plastic polycarbonate, Canada). The chemicals used for media preparations were of analytical grade (Duchefa Biochemie, Haarlem, The Netherlands and Sigma Aldrich, Inc., St Louis, USA).
- the radical tissues also started to bulge within two weeks. These radical tissues were completely removed from the explants. Sub-culturing was carried out at two week intervals. After six weeks of culture in stage 1 medium, the SCPC (FW 0.5g) were injured and transferred to stage 2 medium for a short duration of 5 days with subsequent transfer to stage 3 medium for ten days. During this period, shoot buds started to induce from the plumular callus as well as in and around the cotyledonary node ( Figure 1C). Simultaneously, enormous phenolic exudates started oozing out from the base of the shoot buds.
- stage 2 medium In order to avoid these phenolic exudates, Asc, PVP, PVPP, glycine and AgNO 3 were added to stage 2 medium. Based on our observations, long time culture of SCPC more than five days in stage 2 medium inhibited the developing shoot buds which simultaneously, became brown and ultimately senesced. This could be due to PVPP and AgNO 3 acting as inhibitors of the tissue growth when cultured for a long time. To avoid this browning and senescing, subculture was done at five days interval. Our observations coincide with those of Zhao et al. (2000) who have reported the negative impacts of associated phenolic compound production from sorghum tissue culture and can be overcome by short subculture intervals and the addition of PVPP to tissue culture media. In contrast, Carvalho et al.
- auxin (2,4-D) concentration was reduced, cytokinins, BA, KN and organic additives such as CH, AdSO 4 , PVP were added to enhance shoot bud proliferation and growth.
- organic additives such as CH, AdSO 4 , PVP were added to enhance shoot bud proliferation and growth.
- PRGs Plant growth regulators
- PVP 5 CH, L-glutamine organic additives
- auxin was essential for root induction and without auxin, there was no root formation. An average of 10-15 roots of 3 cm long were produced per shoot when cultured in stage 6 medium. The rooted plantlets were successfully hardened with the (100 %) survivability and transferred to the green house ( Figure II).
- Cai et al. (1987) have developed organogenic system from shoot portions of mature embryo in three stages, stage 1 modified MS medium with higher concentration of 2,4-D and lower concentration of KN and in stage 2, MS medium with lower concentration of 2,4-D and higher concentration of KN for callus induction followed by stage 3, MS medium with lower concentration of KN with or without ⁇ -naphthalene acetic acid (NAA) for regeneration of plantlets.
- stage 1 modified MS medium with higher concentration of 2,4-D and lower concentration of KN and in stage 2
- MS medium with lower concentration of 2,4-D and higher concentration of KN for callus induction followed by stage 3
- MS medium with lower concentration of KN with or without ⁇ -naphthalene acetic acid (NAA) for regeneration of plantlets.
- amino acid L-asparagine was essential for the regeneration system and the efficiency of plant regeneration is around 11 -8% and over all, 700 plants were obtained after a prolonged period of nine months.
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PCT/SG2008/000266 WO2010011175A1 (en) | 2008-07-23 | 2008-07-23 | Method of high frequency regeneration of sorghum |
AU2008359743A AU2008359743B2 (en) | 2008-07-23 | 2008-07-23 | Method of high frequency regeneration of sorghum |
BRPI0822966-0A BRPI0822966B1 (en) | 2008-07-23 | 2008-07-23 | high frequency sorghum regeneration method |
CN2008801304933A CN102105048B (en) | 2008-07-23 | 2008-07-23 | Method of high frequency regeneration of sorghum |
US13/003,971 US8431402B2 (en) | 2008-07-23 | 2008-07-23 | Method of high frequency regeneration of Sorghum |
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JP2019520820A (en) * | 2016-06-10 | 2019-07-25 | コモンウェルス サイエンティフィック アンド インダストリアル リサーチ オーガナイゼーション | How to improve sorghum gene transformation |
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WO2015099674A1 (en) * | 2013-12-23 | 2015-07-02 | Monsanto Technology Llc | Sugarcane regeneration and transformation methods |
CN104686371B (en) * | 2015-04-04 | 2016-11-30 | 青岛玉兰祥商务服务有限公司 | A kind of Sorghum vulgare Pers. flower pesticide inducing culture formula |
CN110663547B (en) * | 2018-07-03 | 2022-04-08 | 中国科学院分子植物科学卓越创新中心 | Culture method of cryptomeria draconis and picea mucronata explants |
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EP0176162A1 (en) * | 1984-09-07 | 1986-04-02 | Sungene Technologies Corporation | Process for regenerating cereals |
WO2001067861A1 (en) * | 2000-03-10 | 2001-09-20 | Invitrogen Corporation | Materials and methods for the regeneration of plants from cultured plant tissue |
WO2002014520A2 (en) * | 2000-08-17 | 2002-02-21 | Her Majesty The Queen In Right Of Canada As Represented By The Minister Of Agriculture And Agri-Food | Process for inducing direct somatic embryogenesis and secondary embryogenesis in monocotyledonous plant cells, and rapidly regenerating fertile plants |
WO2005029944A2 (en) * | 2003-09-22 | 2005-04-07 | J.R. Simplot Company | Refined plant transformation |
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CN101218893B (en) * | 2007-12-29 | 2011-12-28 | 天津农学院 | Method for evoking sorghum mature embryo callus and establishing regeneration system |
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EP0176162A1 (en) * | 1984-09-07 | 1986-04-02 | Sungene Technologies Corporation | Process for regenerating cereals |
WO2001067861A1 (en) * | 2000-03-10 | 2001-09-20 | Invitrogen Corporation | Materials and methods for the regeneration of plants from cultured plant tissue |
WO2002014520A2 (en) * | 2000-08-17 | 2002-02-21 | Her Majesty The Queen In Right Of Canada As Represented By The Minister Of Agriculture And Agri-Food | Process for inducing direct somatic embryogenesis and secondary embryogenesis in monocotyledonous plant cells, and rapidly regenerating fertile plants |
WO2005029944A2 (en) * | 2003-09-22 | 2005-04-07 | J.R. Simplot Company | Refined plant transformation |
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Cited By (4)
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JP2019520820A (en) * | 2016-06-10 | 2019-07-25 | コモンウェルス サイエンティフィック アンド インダストリアル リサーチ オーガナイゼーション | How to improve sorghum gene transformation |
EP3469068A4 (en) * | 2016-06-10 | 2019-11-06 | Commonwealth Scientific and Industrial Research Organisation | Methods to improve genetic transformation of sorghum |
JP7250524B2 (en) | 2016-06-10 | 2023-04-03 | コモンウェルス サイエンティフィック アンド インダストリアル リサーチ オーガナイゼーション | Methods for improving genetic transformation of sorghum |
US11917960B2 (en) | 2016-06-10 | 2024-03-05 | Commonwealth Scientific And Industrial Research Organisation | Methods to improve genetic transformation of sorghum |
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